Members of the genus Nocardia, which are partially acid-fast, aerobic, branched gram-positive bacilli, are opportunistic pathogens commonly found in patients with acute or chronic, suppurative or granulomatous diseases (McNeil and Brown (1994) Clin. Microbiol. Rev. 7: 357-417). Of the greatest clinical importance within this genus is Nocardia farcinica, a member of the N. asteroides complex composed of Nocardia abscessus, Nocardia asteroides sensu stricto type VI (Nocardia cyriacigeorgica), Nocardia nova, and Nocardia farcinica (Wallace et al., (1988) Antimicrob. Agents Chemother. 32:1776-1779). Nocardia farcinica causes localized and disseminated infections, predominantly affecting immunocompromised patients. Differentiation of Nocardia farcinica from other members of N. asteroides complex is important because Nocardia farcinica has a high degree of resistance to various antibiotics, especially to the third-generation cephalosporins, which may make it difficult to treat (Wallace et al., (1988) Antimicrob. Agents Chemother. 32:1776-1779; Wallace et al., (1990) J. Clin. Microbiol 28:2726-2732) and because mouse pathogenicity studies have demonstrated that it may be more virulent than the other N. asteroides complex species (Desmond and Flores (1993) FEMS Microbiol. Lett. 110: 281-284).
Human and animal clinical infections with Nocardia farcinica may occur more frequently than previously recognized (Cohen et al., (2000) Eur. J. Clin. Microbiol. Infect. Dis. 19: 224-227; Manninen et al., (1993) Can. J. Microbiol. 39: 635-641; Torres et al., Eur. J. Clin. Microbiol Infect. Dis. 19: 205-212). Other reports from France, Germany, and the United States have implicated Nocardia farcinica as the cause of postoperative wound infections in patients undergoing cardiac and other vascular surgeries (Blumel et al., (1998) J. Clin. Microbiol. 36:118-122; Boiron et al., (1998) Med. Mycol. 36 Suppl 1: 26-37; Exmelin et al., J. Clin. Microbiol. 34: 1014-1016; Wenger et al., (1998) J. Infect. Dis. 178: 1539-1543). From 1987 through 1989, one of the largest known nocardial mastitis epizootics was reported in all 10 Canadian provinces (Manninen et al., (1993) Can. J. Microbiol. 39: 635-641). The causative agent of the outbreak was initially reported as Nocardia species, but later was presumptively identified as Nocardia farcinica (Manninen et al., (1993) Can. J. Microbiol. 39: 635-641). Further phenotypic and molecular testing at the Actinomycete Reference Laboratory confirmed the identification. It is important to rapidly identify the pathogen not only for antimicrobial therapeutic purposes, but also to establish the spectra of disease and to monitor and control the rate of infection.
Traditional biochemical identification of Nocardia farcinica is often laborious, difficult to replicate, and time-consuming; species identification usually requires up to 3 weeks. In addition, misidentification of Nocardia farcinica may occur because of the phenotypic similarity to species in the related genera, Gordonia, Rhodococcus, and rapidly growing Mycobacterium. Commercially available systems in combination with a few traditional tests have shortened the identification time of Nocardia species to 7 days; however, phenotypic identification to the species level within this genus remains problematic (Biehle et al. (1996) J. Clin. Microbiol. 34: 103-107; Kiska et al., (2002) J. Clin. Microbiol. 40:1346-1351; Muir and Pritchard (1997) J. Clin. Microbiol. 35: 3240-3233). For example, within the genus Nocardia are recently described species, Nocardia africana, Nocardia vaccinii, and Nocardia veterana, that share similar phenotypic (biochemical and susceptibility profiles) and molecular characteristics to N. nova (Conville et al., (2003) J. Clin. Microbiol. 41: 2560-2568). The use of molecular approaches such as PCR targeting portions of the hsp gene and the 16S rRNA gene coupled with restriction endonuclease digestion of PCR products has been the focus of recent investigations for the separation of mycobacteria from the nocardiae as well as for the recognition of species within the genera Mycobacterium and Nocardia (Conville et al., (2003) J. Clin. Microbiol. 41: 2560-2568; Conville et al., (2000) J. Clin. Microbiol. 38: 158-164; Laurent et al., (1999) J. Clin. Microbiol. 37: 99-102); Lungu et al., (1994) Diagn. Microbiol. Infect. Dis. 18: 13-18; Steingrube et al., (1995) J. Clin. Microbiol. 33: 3096-3101; Wilson et al., (1998) J. Clin. Microbiol. 36:148-152). Such methodology has proven to be sensitive, less time-consuming, and less labor-intensive than traditional biochemical methods. However, accurate identification may still be difficult because it relies upon analysis of a relatively few restriction fragments from a single gene, many of which co-migrate between different species. Recently, randomly amplified polymorphic DNA (RAPD) analysis has been described as an identification method for the Nocardia species (Isik and Goodfellow (2002) Syst. Appl. Microbiol. 25:60-67). RAPD analysis has also been described as a useful method for intraspecies discrimination in an epidemiological study of Nocardia farcinica (Exmelin et al., J. Clin. Microbiol. 34: 1014-1016). Exmelin et al. (Exmelin et al., J. Clin. Microbiol. 34: 1014-1016), using RAPD analysis for subtyping, incorporated a single short primer, DKU49, in a low stringency PCR to amplify genomic DNA. While relatively easy to perform, identification based on RAPD profiles suffers from the disadvantage of lacking reproducible profiles unless stringent reaction conditions are observed. For instance, profiles were determined to be dependent on reaction parameters such as annealing temperature, magnesium concentration, the type of thermal cycler, source of DNA polymerase, and both primer and template concentrations. In addition, except for a pronounced 409-bp band, variation of DKU49 profiles were observed among strains of Nocardia farcinica making identification based on RAPD profiles alone difficult (FIG. 1). No correlation between RAPD profiles and epidemiologic relatedness was observed.
To overcome the above disadvantages, compositions and methods to identify Nocardia farcinica are disclosed.